Inactivated and Immunogenic SARS-CoV-2 for Safe Use in Immunoassays and as an Immunization Control for Non-Clinical Trials

Abstract

Successful SARS-CoV-2 inactivation allows its safe use in Biosafety Level 2 facilities, and the use of the whole viral particle helps in the development of analytical methods and a more reliable immune response, contributing to the development and improvement of in vitro and in vivo assays. In order to obtain a functional product, we evaluated several inactivation protocols and observed that 0.03% beta-propiolactone for 24 h was the best condition tested, as it promoted SARS-CoV-2 inactivation above 99.99% and no cytopathic effect was visualized after five serial passages. Moreover, RT-qPCR and transmission electron microscopy revealed that RNA quantification and viral structure integrity were preserved. The antigenicity of inactivated SARS-CoV-2 was confirmed by ELISA using different Spike-neutralizing monoclonal antibodies. K18-hACE2 mice immunized with inactivated SARS-CoV-2, formulated in AddaS03TM, presented high neutralizing antibody titers, no significant weight loss, and longer survival than controls from a lethal challenge, despite RNA detection in the oropharyngeal swab, lung, and brain. This work emphasizes the importance of using different techniques to confirm viral inactivation and avoid potentially disastrous contamination. We believe that an efficiently inactivated product can be used in several applications, including the development and improvement of molecular diagnostic kits, as an antigen for antibody production as well as a control for non-clinical trials.

Influenza Virus Inactivated by Heavy Ion Beam Irradiation Stimulates Antigen-Specific Immune Responses

“…The use of irradiation will also render dispensable cumbersome approaches to remove potential residual toxic moieties [ 31 , 51 ]. Thus, during the SARS-CoV-2 pandemic, several vaccine candidates inactivated using irradiation technologies were tested [ 26 , 52 , 53 , 54 , 55 ]. In addition, also from a commercial point of view, technologies such as electron-based beams (eBeam) or X-ray seem to be attractive tools due to the overall lower costs.…”

Plaque Reduction Neutralization Test (PRNT) Accuracy in Evaluating Humoral Immune Response to SARS-CoV-2

“…SARS-CoV-2 viruses (Wuhan and Omicron) were kindly provided by the Laboratory of Respiratory Viruses, Exanthematics, Enteroviruses and Viral Emergencies (LVRE) at IOC/Fiocruz (SISGEN A994A37). Virus propagation (Wuhan strain, 10.10 log 5 PFU/mL; Omicron variant, 9.32 log 5 PFU/mL) was carried out in Vero E6 cells (CRL-1586, ATCC, Manassas, VA, USA) maintained in 199 medium with Earle salts (E199, Sigma, Livonia, MI, USA) buffered with sodium bicarbonate and supplemented with 5% fetal bovine serum (FBS, Invitrogen, Waltham, MA, USA), as described previously [34]. The African green monkey Vero CCL81 (ATCC, Manassas, VA, USA) cells were cultivated in 24-well plates (120,000 or 200,000 cells/well) one day before the assay (or two days before, in the case of robustness assay for validation step).…”